Combination of Broadly Neutralizing HIV Antibodies and Viral Inducers

ABSTRACT

The present invention relates to methods and agents for preventing the establishment of HIV-1 latent reservoirs or for reducing the size of the reservoirs. Specifically, the disclosure provides methods and agents for preventing the establishment of HIV-1 latent reservoirs or for reducing the size of the reservoirs, the methods comprising administering to the subject a therapeutically effective amount of an isolated anti-HIV antibody, and administering to the subject two or more viral transcription inducers in effective amounts to induce transcription of an HIV provirus in the cells. Further provided are antibodies and viral transcription inducers used in the methods.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/026,915 filed on Jul. 21, 2014. The content of the application isincorporated herein by reference in its entirety.

GOVERNMENT INTERESTS

The invention disclosed herein was made, at least in part, withGovernment support under Grant Nos. T32GM07739, AI100663-02,AI100148-02, AI081677-05, and #8 UL1 TR000043 from the NationalInstitutes of Health (NIH). Accordingly, the U.S. Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates to methods and agents for prevention ordisruption of the establishment or maintenance of human immunodeficiencyvirus latent reservoirs.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is a retrovirus that causes acquiredimmunodeficiency syndrome (AIDS). HIV-1 is the most common andpathogenic strain of the virus. Although HIV-1 infection can besuppressed with any one of several different combination anti-retroviraltherapies (ART), such a therapy must be maintained for the life of apatient because it does not eliminate a reservoir of latently infectedcells even after years of ART (Siliciano, et al., Nat Med, 2003. 9(6):pp. 727-8.). As a result, ART termination follows by rapid viral rebound(Davey, et al., Proc. Natl. Acad. Sci. U.S.A. 1999. pp. 15109-15114). Todate, all attempts to alter the reservoir by intensifying ART, byincluding additional anti-retroviral drugs (Dinoso, et al., Proc NatlAcad Sci U.S.A., 2009. 106(23) pp. 9403-8 and Gandhi, et al., PLoS Med,2010. 7(8)), or by administration of global T-cell activators orinducers of viral transcription, in the presence of ART, have failed(Archin, et al., J. Infect. Dis. 2014. pp. 1-26; Dybul, et al., InfectDis, 2002. 185(1): pp. 61-8; Lafeuillade, et al., J Acquir Immune DeficSyndr, 2001. 26(1): p. 44-55; and Prins, et al., AIDS, 1999. 13(17): p.2405-10). Thus, there is a need for methods and agents for prevention ordisruption of the establishment or maintenance of HIV-1 latentreservoirs.

SUMMARY OF INVENTION

This invention relates to using broadly neutralizing antibodies (bNAbs)alone or in combination with viral transcription inducers in preventingthe establishment of the latent reservoirs of HIV-1 infected cells ordecreasing the size of the reservoir, and thereby addresses the needmentioned above.

Accordingly, one aspect of this invention provides a method fordecreasing the size of or preventing the establishment of a latentreservoir of HIV infected cells (e.g., a cell population comprisingHIV-infected CD4⁺ T cells.) in a subject in need thereof. The methodincludes administering to the subject a therapeutically effective amountof an isolated anti-HIV antibody, e.g., a bNAb, and administering to thesubject two or more viral transcription inducers in effective amounts toinduce transcription of an HIV provirus in the cells.

The antibody can be a human antibody, a humanized antibody, or achimeric antibody. In one example, the antibody is antibody 3BNC117,10-1074, or PG16, or any other described below, or combination thereof.Preferably, two or three of the antibodies 3BNC117, 10-1074, and PG16are administered to the subject. The antibody or antibodies can beadministered to the subject within about 96 hours or earlier (e.g., 72,48, 36, 24, or 12 hours) after exposure or suspected exposure to HIV.The transcription inducers can be administered to the subject for aperiod of about 5-14 days (e.g., 6-13, 7-12, 8, 9, 10, 11, 12 days).Examples of the transcription inducers include vorinostat, an HDACinhibitor, I-BET151, a BET bromodomain inhibitor, and αCTLA4, a T-cellinhibitory pathway blocker. The method can further compriseadministering to the subject an antiviral agent, such as one selectedfrom the group consisting of a non-nucleoside reverse transcriptaseinhibitor, a protease inhibitor, an entry or fusion inhibitor, and anintegrase inhibitor.

In a second aspect, the invention provides a kit comprising an isolatedanti-HIV antibody, a first viral transcription inducer, and a secondviral transcription inducer. The kit can contain one, two, three, ormore of antibodies 3BNC117, 10-1074, PG16, and those described below.The antibody can be a human antibody, a humanized antibody, or achimeric antibody. The transcription inducers can be selected from thegroup consisting of vorinostat, an HDAC inhibitor, I-BET151, a BETbromodomain inhibitor, and αCTLA4, a T-cell inhibitory pathway blocker.In the kit, the first viral transcription inducer and the second viraltranscription inducer can be contained in one pharmaceutical compositionor in two separate pharmaceutical compositions. The kit can furthercontain an antiviral agent, which can be selected from the groupconsisting of a non-nucleoside reverse transcriptase inhibitor, aprotease inhibitor, an entry or fusion inhibitor, and an integraseinhibitor. The kit can be used in the method of this invention.

In a third aspect, the invention features a method for preventing theestablishment of a latent reservoir of HIV infected cells in a subjectin need thereof. The method includes administering to the subject atherapeutically effective amount of an isolated anti-HIV antibody. Theantibody can be a human antibody, a humanized antibody, or a chimericantibody. In one example, the antibody is antibody 3BNC117, 10-1074,PG16, any of those described below, or a combination thereof.Preferably, two, three, or more of the antibodies are administered tothe subject. The antibody or antibodies can be administered to thesubject within about 96 hours or earlier (e.g., 72, 48, 36, 24, or 12hours) after exposure or suspected exposure to HIV.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objectives, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F, AND FIG. 1H are aset of diagrams showing post-exposure prophylaxis with bNAbs: (FIG. 1A)Schematic timeline for the bNAb (top) and ART (bottom) experiments.(FIG. 1B) Plasma viremia for untreated mice. The x-axis is in days postHIV-1 challenge. The y-axis is viral RNA copies/ml. Gray shadingindicates values beneath the detection limit of 800 copies/ml. The blueline indicates the geometric mean plasma viremia. (FIG. 1C) Plasmaviremia for ART-treated mice as in FIG. 1B. The blue shading indicatesthe treatment period with ART. (FIG. 1D) Plasma viremia forantibody-treated mice. The red arrows indicate antibody tri-mixinjections. The dashed red line shows average plasma antibodyconcentration for all mice in the group. (FIG. 1E) Graph as in FIG. 1D,for mice treated with antibody starting 8 days after HIV-1 challenge.(FIG. 1F) Percentage of CD4⁺ T cells in the spleen at the terminal pointmeasured by flow cytometry, organized by treatment group. (A=aviremic,V=viremic). (FIG. 1G) Cell-associated HIV-1 RNA measured in spleen Tcells at the terminal point, plotted as the ratio of HIV-1 RNA to CCR5copies for each mouse. Gray shading indicates the detection limit of1.25×10⁻⁵ copies per cell. (FIG. 1H) Cell-associated HIV-1 DNA measuredin spleen T cells at the terminal point, plotted as the ratio of HIV-1RNA to CCR5 copies for each mouse. Gray shading indicates the detectionlimit of 2.0×10⁻⁵ copies per cell.

FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D are a set of diagrams showingthat Fcr^(null) antibodies suppress viremia but do not prevent rebound:(FIG. 2A) Plasma viremia as in FIG. 1D for mice treated with Fcr^(null)tri-mix. (FIG. 2B) The proportion of mice that were viremic at theterminal point for each treatment group (*, p<0.05; Fisher's Exacttest). (FIG. 2C) For all viremic mice, plasma antibody concentration onthe day of viral rebound. Antibody levels were significantly higher inFcr^(null) tri-mix treated mice compared to wild-type tri-mix treatedmice (*, p<0.05; **, p<0.01; Mann-Whitney test). (FIG. 2D) Sequences ofgp120 cloned from plasma. Horizontal lines denote individual clones,grouped by mouse, shown on the right. Red ticks and green ticks indicatenon-synonymous and synonymous substitutions relative to gp120_(YU2),respectively. Blue shading highlights sites of mutations that conferescape to the antibody tri-mix.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E are a set of diagramsshowing rebound viremia after therapy with single inducers: (FIG. 3A)Schematic timeline of the experiment. (FIG. 3B, FIG. 3C, and FIG. 3D)Graphs show plasma viremia for individual mice on the left y-axis,geometric mean antibody level on the right y-axis among all mice in thegroup (red). The x-axis represents days relative to the first antibodyinjection. Antibody injections are indicated with red arrows. Mice thathad rebound plasma viremia are shown in gray. Mice that failed torebound are shown in black. (FIG. 3B) Mice that received tri-mixantibodies, but no inducers. (FIG. 3C) Mice that received tri-mixantibodies and vorinostat (green arrows). (FIG. 3D) Mice that receivedtri-mix antibodies and I-BET151 (purple shading). (FIG. 3E) Mice thatreceived tri-mix antibodies and αCTLA4 (orange arrows).

FIG. 4A and FIG. 4B are a set of diagrams showing that combinationinducers decrease the incidence of rebound viremia: (FIG. 4A) Micetreated with tri-mix of antibodies, and a combination of three inducers.Graph, arrows, and shading are as in FIG. 3. (FIG. 4B) Graph shows theproportion of mice that showed rebound viremia for each treatment group,where all mice that received antibody tri-mix and any one of the threesingle inducers (shown in FIGS. 3C, 3D, and 3E) are pooled together (*,p<0.05; Mann-Whitney test).

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G are aset of diagrams showing antibody persistence and premature terminationdo not account for non-rebounding: (FIG. 5A) Percentage of CD4⁺ T cellsat the terminal point measured in the spleen by flow cytometry. (FIG.5B) Cell-associated HIV-1 RNA measured in spleen cells at terminalpoint, plotted as the ratio of HIV-1 RNA to CCR5 DNA copies for eachmouse. Mice that had measurable HIV-1 RNA, but undetectable CCR5 DNA areplotted as 10⁴ copies per cell. (FIG. 5C) Plasma viremia before therapywas initiated for each mouse, organized by treatment group and reboundstatus (N.R.=non-rebounder, Reb.=viral rebounder). There was nosignificant difference for any individual group (Kruskal-Wallis test).(FIG. 5D) The plasma antibody level at the time of viral rebound foreach mouse that rebounded, organized by treatment group. The mean plasmaantibody level at the time of rebound was 2.97 μg/ml for all groups.(FIG. 5E) For each mouse that rebounded, the number of days that elapsedfrom when the antibody level dropped below 2.97 μg/ml to the time ofrebound. (FIG. 5F) For mice that did not rebound, the number of daysthat elapsed from when each mouse's antibody levels dropped below 2.97μg/ml to the terminal point. (FIG. 5G) Cell-associated HIV-1 DNAmeasured in spleen T cells at the terminal point, plotted as the ratioof HIV-1 DNA to CCR5 copies for each mouse. Mice that had measurableHIV-1 DNA, but undetectable CCR5 DNA are plotted as 10⁴ copies per cell.

FIG. 6A and FIG. 6B are set of diagrams showing viremia and antibodylevels in individual mice (bNAb Post-exposure prophylaxis) as in FIG.1D: (FIG. 6A) Each mouse shown in FIG. 1D is shown individually. (FIG.6B) Each mouse shown in FIG. 1E is shown individually.

FIG. 7 is a set of diagram showing viremia and antibody levels inindividual mice (Fcr^(null) tri-mix Post-exposure prophylaxis) as inFIG. 1D. Each mouse shown in FIG. 2A is shown individually.

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D are a set of diagram showingviremia and antibody levels in individual mice (antibody only andantibody plus single inducers) in FIGS. 3A-3D: (FIG. 8A) Each mouseshown in FIG. 3A is shown individually. (FIG. 8B) Each mouse shown inFIG. 3B is shown individually. (FIG. 8C) Each mouse shown in FIG. 3C isshown individually. (FIG. 8D) Each mouse shown in FIG. 3D is shownindividually.

FIG. 9 is a set of diagram showing viremia in individual mice (antibodyplus combination inducers) as in FIG. 4A. Each mouse shown in FIG. 4A isshown individually.

FIG. 10A, FIG. 10B, and FIG. 10 are a set of diagram showing sustainedviremia in inducers treated mice without therapy: (FIG. 10A) Graphsshown as in FIG. 4A. The blue line indicates the geometric mean viremiaacross all mice. (FIG. 10B) Proportion of human CD45⁺ leukocytes inperipheral blood, measured by flow cytometry. (FIG. 10C) Percentage ofCD4⁺ T cells in the peripheral blood measured by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, on unexpected discoveries (i)that bNAbs are more effective than ART in preventing the establishmentof the latent reservoirs of HIV-1 infected cells by a mechanism thatrequires Fc receptor function and (ii) that bNAbs in combination withviral transcription inducers that act by independent mechanismssynergize to decrease the size of the reservoir as measured byprevention of viral rebound. Thus, combinations of viral transcriptioninducers and bNAbs constitute a therapeutic strategy that impacts theestablishment and maintenance of the HIV-1 reservoir.

A. HIV-1 Latent Reservoir

HIV-1 viruses are transmitted as single-stranded, positive-sense,enveloped RNA viruses. Upon entry into a target host cell, the viral RNAgenome is reverse transcribed into double-stranded DNA by a virallyencoded reverse transcriptase that is transported along with the viralgenome in the virus particle. The resulting viral DNA is then importedinto the cell nucleus and integrated into the cellular DNA by a virallyencoded integrase and host co-factors. Once integrated, the virus maybecome latent, allowing the virus and its host cell to avoid detectionby the immune system. HIV-1 persists in infected individuals in a stablepool or reservoir of resting CD4⁺ T cells and other cells as a latentbut replication-competent provirus. Accordingly, as used herein, theterm “latent reservoir” refers to cells or sites in a host that arelatently infected with a microbe (e.g., HIV). Its presence and size of aHIV latent reservoir can be measured or assessed by viral rebound afterterminating therapy as disclosed in the examples below.

Such a latent reservoir of HIV-1 infected cells cannot be cleared bycombined ART and remains the major barrier to curing HIV-1 infection. Inhumans, macaques and humanized mice, the latent reservoir is establishedwithin days of initial infection and persists for the lifetime of theindividual, making ART a lifelong necessity. While the latentreservoir's exact cellular composition is debated, it is generallybelieved that it consists primarily of CD4⁺ memory T cells harboringreplication competent provirus that is transcriptionally silenced.Because these cells have very long half-lives and may be able to undergohomeostatic proliferation, a “shock and kill” approach has been proposedto eradicate this reservoir by combining ART with inducers of viraltranscription. However, all attempts at altering the HIV-1 reservoirhave failed to date.

As disclosed herein, bNAbs can be used in preventing the establishmentof the reservoir by a mechanism that requires Fc receptor function. Inestablished infection bNAbs plus single inducers are ineffective indecreasing the reservoir size. Surprisingly, bNAbs plus a combination ofinducers that act by independent mechanisms synergize to decrease thesize of the reservoir as measured by prevention of viral rebound. Thus,combinations of inducers and bNAbs constitute a therapeutic strategythat can be used to prevent or disrupt the establishment and maintenanceof the HIV-1 reservoir in humans and related animal models.

B. Antibodies

The invention disclosed herein involves broadly neutralizing HIV-1antibodies. These antibodies refer to a class of neutralizing antibodiesthat neutralize multiple HIV-1 viral strains. Various bNAbs are known inthe art and can be used in this invention. Examples include but are notlimited to those described in U.S. Pat. No. 8,673,307, WO 2014063059,WO2012158948, and U.S. Provisional Application 61/934,359, includingantibodies 3BNC117, 3BNC60, 12A12, 12A21, NIH45-46, bANC131, 8ANC134,IB2530, INC9, 8ANC195. 8ANC196, 10-259, 10-303, 10-410, 10-847, 10-996,10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM.Additional examples include those described in Klein et al., Nature,2012. 492(7427): p. 118-22, Horwitz et al., Proc Natl Acad Sci USA,2013. 110(41): p. 16538-43, Scheid, et al. 2011. Science, 333:1633-1637,Scheid, et al. 2009. Nature, 458:636-640, Eroshkin et al., Nucleic AcidsRes. 2014 January; 42 (Database issue):D1133-9, Mascola et al. ImmunolRev. 2013 July; 254(1):225-44, such as those listed below.

TABLE 1 Antibody binding Viral Epitope characteristics Antibody clonalfamily MPER of gp41 Contiguous sequence 2F5 Contiguous sequence 4E10Contiguous sequence M66.6 Contiguous sequence CAP206-CH12 Contiguoussequence 10E8 1 V1V2-glycan Peptidoglycan PG9, PG16 PeptidoglycanCH01-04 Peptidoglycan PGT 141-145 Outer domain glycan Glycan only 2G12V3-glycan Peptidoglycan PGT121-123 Peptidoglycan PGT125-131Peptidoglycan PGT135-137 CD4 binding site CDRH3 loop b12 No ligandedstructure HJ16 CDRH3 loop CH103-106 Mimics CD4 via CDRH2 VRC01-03 MimicsCD4 via CDRH2 VRC-PG04, 04b Mimics CD4 via CDRH2 VRC-CH30-34 No ligandedstructure 3BNC117, 3BNC60 Mimics CD4 via CDRH2 NIH45-46 No ligandedstructure 12A12, 12A21 No liganded structure 8ANC131, 134 No ligandedstructure 1NC9, 1B2530

Listed below are the heavy chain variable regions (HC) sequences andlight chain variable regions (LC) of some of the antibodies mentionedabove, where the CDRs are in bold.

Antibody Sequence SEQ ID NO: 3BNC117HCQVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAP  1GQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVWGSGTQVTVSS ASTKG 3BNC117kcDIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPK  2LLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPSVFIFPPSD 3BNC60HCQVHLSQSGAAVTKPGASVRVSCEASGYKISDHFIHWWRQAP  3GQGLQWVGWINPKTGQPNNPRQFQGRVSLTRQASWDFDTYSFYMDLKAVRSDDTAIYFCARQRSDFWDFDVWGSGTQVTVSS ASTKG 3BNC60KCDIQMTQSPSSLSARVGDTVTITCQANGYLNWYQQRRGKAPK  4LLIYDGSKLERGVPARFSGRRWGQEYNLTINNLQPEDVATY FCQVYEFIVPGTRLDLKRTVAA 12A12HCSQQLVQSGTQVKKPGASVRISCQASGYSFTDYVLHWWRQAP  5GQGLEWMGWIKPVYGARNYARRFQGRINFDRDIYREIAFMDLSGLRSDDTALYFCARDGSGDDTSWHLDPWGQGTLVIVSAA STKG 12a12kcDIQMTQSPSSLSASVGDRVTITCQAGQGIGSSLQWYQQKPG  6KAPKLLVHGASNLHRGVPSRFSGSGFHTTFSLTISGLQRDDFATYFCAVLEFFGPGTKVEIKRTVAAPSVFIFPPSDEQLKS 12A21HCSQHLVQSGTQVKKPGASVRVSCQASGYTFTNYILHWWRQAP  7GQGLEWMGLIKPVFGAVNYARQFQGRIQLTRDIYREIAFLDLSGLRSDDTAVYYCARDESGDDLKWHLHPWGQGTQVIVSPA STKG 12a21kcDIQMTQSPSSLSASVGDRVTINCQAGQGIGSSLNWYQKKPG  8RAPKLLVHGASNLQRGVPSRFSGSGFHTTFTLTISSLQPDDVATYFCAVFQWFGPGTKVDIKRTVAAPSVFIFPPSDEQLK NIH-45-QVRLSQSGGQMKKPGESMRLSCRASGYEFLNCPINWIRLAP  9 46HCGRRPEWMGWLKPRGGAVNYARKFQGRVTMTRDVYSDTAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEH NIH-45-EIVLTQSPATLSLSPGETAIISCRTSQSGSLAWYQQRPGQA 10 46kCPRLVIYSGSTRAAGIPDRFSGSRWGADYNLSISNLESGDFG VYYCQQYEF 8ANC131HCQGQLVQSGGGLKKPGTSVTISCLASEYTFNEFVIHWIRQAP 11GQGPLWLGLIKRSGRLMTAYNFQDRLRLRRDRSTGTVFMELRGLRPDDTAVYYCARDGLGEVAPDYRYGIDVWGQGSTVIVT AASTKG 8ANC131KCEIVLTQSPATLSLSPGERATLSCRASQGLNFVVWYQQKRGQ 12APRLLIHAPSGRAPGVPDRFSARGSGTEFSLVISSVEPDDFAIYYCQEYSSTPYNFGPGTRVDRKRTVAAPSVFIFPPSDEQ 8ANC134HCQGQLVQSGGGVKKPGTSVTISCLASEYTFNEFVIHWIRQAP 13GQGPVWLGLIKRSGRLMTSYKFQDRLSLRRDRSTGTVFMELRGLRLDDTAVYYCARDGLGEVAPAYLYGIDAWGQGSTVIVT SASTKG 8ANC134KCEIVLTQSPATLSLSPGERATLSCRASQGLNFVVWYQQKGGQ 14APRLLIHGPTDRAPGVPDRFSARGSGTEFSLVISSVEPDDFALYYCQEYSSTPYNFGPGTRVDRKRTVAAPSVFIFPPSDEQ IB2530HCQVQLEQSGTAVRKPGASVTLSCQASGYNFVKYIIHWVRQKP 15GLGFEWVGMIDPYRGRPWSAHKFQGRLSLSRDTSMEILYMTLTSLKSDDTATYFCARAEAASDSHSRPIMFDH B2530KCQVQLEQSGTAVRKPGASVTLSCQASGYNFVKYIIHWVRQKP 16GLGFEWVGMIDPYRGRPWSAHKFQGRLSLSRDTSMEILYMTLTSLKSDDTATYFCARAEAASDSHSRPIMFDH INC9HCQVQLEQSGTAVRKPGASVTLSCQASGYNFVKYIIHWVRQKP 17GLGFEWVGMIDPYRGRPWSAHKFQGRLSLSRDTSMEILYMTLTSLKSDDTATYFCARAEAASDSHSRPIMFDH INC9KCQVQLEQSGTAVRKPGASVTLSCQASGYNFVKYIIHWVRQKP 18GLGFEWVGMIDPYRGRPWSAHKFQGRLSLSRDTSMEILYMTLTSLKSDDTATYFCARAEAASDSHSRPIMFDH 8ANC195HCQIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVRQA 19PGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTYDKWSGLHHDGVMAFSSWG QGTLISVSAASTKG 8ANC195KCDIQMTQSPSTLAASIGGTVRVSCRASQSITGNWVAWYQQRP 20GKAPRLLIYRGAALLGGVPSRFSGSAAGTDFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKVEVKRTVAAPSVFIFPPSD EQ

The heavy chain variable regions (IgH) and light chain variable regions(IgL) of additional antibodies are listed below, where the CDRs are inbold.

IgH Sequences

IMGT Sequence SEQ ID NO: 10-1369QVQLQESGPGLVKPLETLSLTCNVSGAFIADHYWSWIRLPL 21GKGPEWIGYVHDSGDINYNPSLKNRVHLSLDKSTNQVSLKLMAVTAGDSALYYCATTKHGRRIYGWAFGEWFTYFYMDVWG RGTTVTVSS 10-259QVHLQESGPGLVKPSETLSLTCNVSGTLVRDNYWSWMRQPL 22GKQPEWIGYVHDSGDTNYNPSLKSRVHLSLDKSNNLVSLRLTAVTAADSATYYCATTKHGRRIYGIVAFNEWFTYFYMDVWG KGTTVTVSS 10-303QVQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSP 23GKGLEWIGYVHKSGDTNYSPSLKSRVNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWG NGTQVTVSS 10-410QVQLQESGPGLVKPPETLSLTCSVSGASVNDAYWSWIRQSP 24GKRPEWVGYVHHSGDTNYNPSLKRRVTFSLDTAKNEVSLKLVALTAADSAVYFCARALHGKRIYGIVALGELFTYFYMDVWG KGTTVTVSS 10-1130QVQLQESGPGLVKPPETLSLTCSVSGASINDAYWSWIRQSP 25GKRPEWVGYVHHSGDTNYNPSLKRRVTFSLDTAKNEVSLKLVDLTAADSAVYFCARALHGKRIYGIVALGELFTYFYMDVWG KGTTVTVSS 10-1121QVQLQESGPGLVKPPETLSLTCSVSGASINDAYWSWIRQSP 26GKRPEWVGYVHHSGDTNYNPSLKRRVSFSLDTAKNEVSLKLVDLTAADSAIYFCARALHGKRIYGIVALGELFTYFYMDVWG KGTTVTVSS 10-1146QVQLVESGPGLVTPSETLSLTCTVSNGSVSGRFWSWIRQSP 21GRGLEWIGYFSDTDRSEYSPSLRSRLTLSLDASRNQLSLKLKSVTAADSATYYCARAQQGKRIYGIVSFGEFFYYYYMDAWG KGTAVTVSS 10-996QVQLQESGPGLVKPSETLSLTCSVSNGSVSGRFWSWIRQSP 28GRGLEWIGYFSDTEKSNYNPSLRSRLTLSVDASKNQLSLKLNSVTAADSATYYCARTQQGKRIYGWSFGEFFHYYYMDAWG KGTAVTVSS 10-1341QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP 29GKGLEWIGYISDRESATYNPSLNSRVVISRDTSTNQLSLKLNSVTPADTAVYYCATARRGQRIYGWSFGEFFYYYSMDVWG RGTTVTVSS 10-847QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP 30GKGLEWIGYISDRASATYNPSLNSRWISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGWSFGEFFYYYSMDVWG KGTTVTVSS 10-1074QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP 31GKGLEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGWSFGEFFYYYSMDVWG KGTTVTVSS 10-1074GMQVQLQESGPGLVKPSETLSVTCSVSGDSMNNSYWTWIRQSP 32GKGLEWIGYISKSESANYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARHGQRIYGWSFGEFFTYYSMDVWG KGTTVTVSS KABAT SequenceSEQ ID NO: 10-1369 QVQLQESGPGLVKPLETLSLTCNVSGAFIADHYWSWIRLPL 21GKGPEWIGYVHDSGDINYNPSLKNRVHLSLDKSTNQVSLKLMAVTAGDSALYYCATTKHGRRIYGWAFGEWFTYFYMDV WGRGTTVTVSS 10-259QVHLQESGPGLVKPSETLSLTCNVSGTLVRDNYWSWMRQPL 22GKQPEWIGYVHDSGDTNYNPSLKSRVHLSLDKSNNLVSLRLTAVTAADSATYYCATTKHGRRIYGIVAFNEWFTYFYMDVWG KGTTVTVSS 10-303QVQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSP 23GKGLEWIGYVHKSGDTNYSPSLKSRVNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWG NGTQVTVSS 10-410QVQTQESGPGLVKPPFTLSLTCSVSGASVNDAYWSWIRQSP 24GKRPEWVGYVHHSGDTNYNPSLKRRVTFSLDTAKNEVSLKLVALTAADSAVYFCARALHGKRIYGIVALGELFTYFYMDVWG KGTTVTVSS 10-1130QVQLQESGPGLVKPPETLSLTCSVSGASINDAYWSWIRQSP 25GKRPEWVGYVHHSGDTNYNPSLKRRVTFSLDTAKNEVSLKLVDLTAADSAVYFCARALHGKRIYGIVALGELFTYFYMDVWG KGTTVTVSS 10-1121QVQLQESGPGLVKPPETLSLTCSVSGASINDAYWSWIRQSP 26GKRPEWVGYVHHSGDTNYNPSLKRRVSFSLDTAKNEVSLKLVDLTAADSAIYFCARALHGKRIYGIVALGELFTYFYMDVWG KGTTVTVSS 10-1146QVQLVESGPGLVTPSETLSLTCTVSNGSVSGRFWSWIRQSP 27GRGLEWIGYFSDTDRSEYSPSLRSRLTLSLDASRNQLSLKLKSVTAADSATYYCARAQQGKRIYGIVSFGEFFYYYYMDAWG KGTAVTVSS 10-996QVQLQESGPGLVKPSETLSLTCSVSNGSVSGRFWSWIRQSP 28GRGLEWIGYFSDTEKSNYNPSLRSRLTLSVDASKNQLSLKLNSVTAADSATYYCARTQQGKRIYGVVSFGEFFHYYYMDAWG KGTAVTVSS 10-1341QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP 29GKGLEWIGYISDRESATYNPSLNSRVVISRDTSTNQLSLKLNSVTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWG RGTTVTVSS 10-847QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP 30GKGLEWIGYISDRASATYNPSLNSRWISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWG KGTTVTVSS 10-1074QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP 31GKGLEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWG KGTTVTVSS 10-1074GMQVQLQESGPGLVKPSETLSVTCSVSGDSMNNSYWTWIRQSP 32GKGLEWIGYISKSESANYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARHGQRIYGVVSFGEFFTYYSMDVWG KGTTVTVSS

IgH Sequences

IMGT Sequence SEQ ID NO: 10-1369SSMSVSPGETAKITCGEKSIGSRAVQWYQKKPGQPPSLIIY 33NNQDRPSGVPERFSASPDIEFGTTATLTITNVEAGDEADYY CHIYDARRPTNWVFDRGTTLTVL 10-259SSMSVSPGETAKISCGKESIGSRAVQWYQQKSGQPPSLIIY 34NNQDRPSGVPERFSATPDFGAGTTATLTITNVEADDEADYY CHIYDARGGTNWVFDRGATLTVL 10-303SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIY 35NNQDRPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYY CHIWDSRVPTKWVFGGGTTLTVL10-1121 SFVSVAPGQTARITCGEESLGSRSVIWYQQRPGQAPSLIMY 36NNHDRPSGIPERFSGSPGSTFGTTATLTITSVEAGDEADYY CHIWDSRRPTNWVFGEGTTLTVL 10-410SFVSVAPGQTARITCGEESLGSRSVIWYQQRPGQAPSLIIY 37NNNDRPSGIPERFSGSPGSTFGTTATLTITSVEAGDEADYY CHIWDSRRPTNWVFGEGTTLTVL10-1130 SFVSVAPGQTARITCGEESLGSRSVIWYQQRPGQAPSLIIY 38NNNDRPSGIPERFSGSPGSTFGTTATLTITSVEAGDEADYY CHIWDSRRPTNWVFGEGTTLTVL 10-847SYVRPLSVALGETASISCGRQALGSRAVQWYQHRPGQAPIL 39LIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEA DYYCHMWDSRSGFSWSFGGATRLTVL10-1074 SYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPIL 40 andLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEA 10-1074GMDYYCHMWDSRSGFSWSFGGATRLTVL 10-1341SYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPIL 41LIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEA DYYCHMWDSRSGFSWSFGGATRLTVL10-996 SSLPLSVAPGATAKIACGEKSFASRAVQWYQQKPGQAPVLI 42IYNNQDRPAGVSERFSGTPDVGFGSTATLTISRVEAGDEAD YYCHKWDSRSPLSWVFGGGTQLTVL10-1146 SSLPLSLAPGATAKIPCGEKSRGSRAVQWYQQKPGQAPTLI 43IYNNQDRPAGVSERYSGNPDVAIGVTATLTISRVEAGDEAE YYCHYWDSRSPISWVFGGWTQLTVLKABAT Sequence SEQ ID NO: 10-1369SSMSVSPGETAKITCGEKSIGSRAVQWYQKKPGQPPSLIIY 33NNQDRPSGVPERFSASPDIEFGTTATLTITNVEAGDEADYY CHIYDARRPTNWVFDRGTTLTVL 10-259SSMSVSPGETAKISCGKESIGSRAVQWYQQKSGQPPSLIIY 34NNQDRPSGVPERFSATPDFGAGTTATLTITNVEADDEADYY CHIYDARGGTNWVFDRGATLTVL 10-303SDISVAPGETARTSCGEKSLGSRAVQWYQHRAGQAPSLIIY 35NNQDRPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYY CHIWDSRVPTKWVFGGGTTLTVL10-1121 SFVSVAPGQTARITCGEESLGSRSVIWYQQRPGQAPSLIMY 36NNHDRPSGIPERFSGSPGSTFGTTATLTITSVEAGDEADYY CHIWDSRRPTNWVFGEGTTLTVL 10-410SFVSVAPGQTARITCGEESLGSRSVIWYQQRPGQAPSLIIY 37NNNDRPSGIPERFSGSPGSTFGTTATLTITSVEAGDEADYY CHIWDSRRPTNWVFGEGTTLTVL10-1130 SFVSVAPGQTARITCGEESLGSRSVIWYQQRPGQAPSLIIY 38NNNDRPSGIPERFSGSPGSTFGTTATLTITSVEAGDEADYY CHIWDSRRPTNWVFGEGTTLTVL 10-847SYVRPLSVALGETASISCGRQALGSRAVQWYQHRPGQAPIL 39LIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEA DYYCHMWDSRSGFSWSFGGATRLTVL10-1074 SYVRPLSVALGETARTSCGRQALGSRAVQWYQHRPGQAPIL 40 andLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEA 10-1074GMDYYCHMWDSRSGFSWSFGGATRLTVL 10-1341SYVRPLSVALGETARTSCGRQALGSRAVQWYQHRPGQAPIL 41LIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEA DYYCHMWDSRSGFSWSFGGATRLTVL10-996 SSLPLSVAPGATAKIACGEKSFASRAVQWYQQKPGQAPVLI 42IYNNQDRPAGVSERFSGTPDVGFGSTAILTISRVEAGDEAD YYCHKWDSRSPLSWVFGGGTQLTVL10-1146 SSLPLSLAPGATAKIPCGEKSRGSRAVQWYQQKPGQAPTLI 43IYNNQDRPAGVSERYSGNPDVAIGVTATLTISRVEAGDEAE YYCHYWDSRSPISWVFGGWTQLTVL

Shown below are the amino acid sequences of PG16 heavy chain and lightchain:

PG16 Igγ1 (SEQ ID No: 54) (T)MGWSCIILFLVATATGVHSQEQLVESGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PG16 Igλ2 (SEQ ID No: 55)(T)MGWSCIILFLVATATGSVT QSALTQPASVSGSPGQTITISCNGTSSDVGGFDSVSWYQQSPGKAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKVTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Italic: leader peptides Bold:variable domains Underlined: constant domains

Like ART, bNAbs can completely suppress viremia in HIV-1 infectedhumanized mice (Klein et al., Nature, 2012. 492(7427): p. 118-22 andHorwitz et al., Proc Natl Acad Sci USA, 2013. 110(41): p. 16538-43) andin SHIV infected macaques (Barouch et al., Nature, 2013. 503(7475): p.224-8 and Shingai et al., Nature, 2013. 503(7475): p. 277-80). Similarto humans, termination of bNAb or ART therapy in hu-mice and macaquesresults in robust viral rebound, indicating persistence of afunctionally silent pool of cells harboring replication-competent virus.Moreover, the relative frequency of latent CD4⁺ T cells as measured byex vivo re-activation is similar in ART suppressed hu-mice and humans(Marsden et al., J Virol, 2012. 86(1): p. 339-47 and Denton et al., JVirol, 2012. 86(1): p. 630-4). Therefore, antibodies and ART containHIV-1 infection in hu-mice but also produce a latent reservoir.

Unlike ART however, antibodies can engage the host immune system byvirtue of their Fc effector domains (Nimmerjahn et al., Nat Rev Immunol,2008. 8(1): p. 34-47) and thereby accelerate clearance of cell freevirus (Igarashi et al., Nat Med, 1999. 5(2): p. 211-6), induce antibodydependent cytotoxicity to kill infected cells (Chung et al., Proc NatlAcad Sci USA, 2011. 108(18): p. 7505-10; Sun et al., J Virol, 2011.85(14): p. 6906-12 Bonsignori et al., J Virol, 2012. 86(21): p.11521-32; Jost et al., Annu Rev Immunol, 2013. 31: p. 163-94; Forthal etal., J Virol, 2001. 75(15): p. 6953-61 and Forthal et al., Curr Opin HIVAIDS, 2013. 8(5): p. 393-401) and produce immune complexes that activatedendritic cells to become potent antigen presenting cells (Dhodapkar etal., Proc Natl Acad Sci USA, 2005. 102(8): p. 2910-5). Finally, bNAbscan prevent cell-cell transmission of HIV-1 (Malbec et al., J Exp Med,2013. 210(13): p. 2813-21 and Abela et al., PLoS Pathog, 2012. 8(4): p.e1002634), whereas ART's activity in this regard is still debated (Sigalet al., Nature, 2011. 477(7362): p. 95-8; Schiffner et al., Vaccine,2013. 31(49): p. 5789-97; and Agosto et al., PLoS Pathog, 2014. 10(2):p. e1003982).

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (for example, bispecificantibodies and polyreactive antibodies), and antibody fragments. Thus,the term “antibody” as used in any context within this specification ismeant to include, but not be limited to, any specific binding member,immunoglobulin class and/or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM,IgA, IgD, IgE and IgM); and biologically relevant fragment or specificbinding member thereof, including but not limited to Fab, F(ab′)2, Fv,and scFv (single chain or related entity). It is understood in the artthat an antibody is a glycoprotein comprising at least two heavy (H)chains and two light (L) chains inter-connected by disulfide bonds, oran antigen binding portion thereof. A heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region (CH1,CH2 and CH3). A light chain is comprised of a light chain variableregion (VL) and a light chain constant region (CL). The variable regionsof both the heavy and light chains comprise framework regions (FWR) andcomplementarity determining regions (CDR). The four FWR regions arerelatively conserved while CDR regions (CDR1, CDR2 and CDR3) representhypervariable regions and are arranged from NH2 terminus to the COOHterminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. Thevariable regions of the heavy and light chains contain a binding domainthat interacts with an antigen while, depending of the isotype, theconstant region(s) may mediate the binding of the immunoglobulin to hosttissues or factors.

Also included in the definition of “antibody” as used herein arechimeric antibodies, humanized antibodies, and recombinant antibodies,human antibodies generated from a transgenic non-human animal, as wellas antibodies selected from libraries using enrichment technologiesavailable to the artisan.

The term “variable” refers to the fact that certain segments of thevariable (V) domains differ extensively in sequence among antibodies.The V domain mediates antigen binding and defines specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the 110-amino acid span of the variableregions. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable regions of nativeheavy and light chains each comprise four FRs, largely adopting a betasheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the beta sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see, for example, Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The term “hypervariable region” as used herein refers to the amino acidresidues of an antibody that are responsible for antigen binding. Thehypervariable region generally comprises amino acid residues from a“complementarity determining region” (“CDR”).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The term “polyclonal antibody” refers to preparationsthat include different antibodies directed against differentdeterminants (“epitopes”).

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with, orhomologous to, corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with, orhomologous to, corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, for example, U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies include antibodies having one or more human antigenbinding sequences (for example, CDRs) and containing one or moresequences derived from a non-human antibody, for example, an FR or Cregion sequence. In addition, chimeric antibodies included herein arethose comprising a human variable region antigen binding sequence of oneantibody class or subclass and another sequence, for example, FR or Cregion sequence, derived from another antibody class or subclass.

A “humanized antibody” generally is considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues often arereferred to as “import” residues, which typically are taken from an“import” variable region. Humanization may be performed following themethod of Winter and co-workers (see, for example, Jones et al., Nature321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting importhypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (see, for example, U.S. Pat. No. 4,816,567), wheresubstantially less than an intact human variable region has beensubstituted by the corresponding sequence from a non-human species.

An “antibody fragment” comprises a portion of an intact antibody, suchas the antigen binding or variable region of the intact antibody.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see, forexample, U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This fragment contains adimer of one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (three loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable region (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” (“sFv” or “scFv”) are antibody fragments that comprisethe VH and VL antibody domains connected into a single polypeptidechain. The sFv polypeptide can further comprise a polypeptide linkerbetween the VH and VL domains that enables the sFv to form the desiredstructure for antigen binding. For a review of sFv, see, for example,Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments with short linkers (about 5-10 residues)between the VH and VL domains such that inter-chain but not intra-chainpairing of the V domains is achieved, resulting in a bivalent fragment,i.e., fragment having two antigen-binding sites. Bispecific diabodiesare heterodimers of two “crossover” sFv fragments in which the VH and VLdomains of the two antibodies are present on different polypeptidechains. Diabodies are described more fully in, for example, EP 404,097;WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993).

Domain antibodies (dAbs), which can be produced in fully human form, arethe smallest known antigen-binding fragments of antibodies, ranging fromabout 11 kDa to about 15 kDa. DAbs are the robust variable regions ofthe heavy and light chains of immunoglobulins (VH and VL, respectively).They are highly expressed in microbial cell culture, show favorablebiophysical properties including, for example, but not limited to,solubility and temperature stability, and are well suited to selectionand affinity maturation by in vitro selection systems such as, forexample, phage display. DAbs are bioactive as monomers and, owing totheir small size and inherent stability, can be formatted into largermolecules to create drugs with prolonged serum half-lives or otherpharmacological activities. Examples of this technology have beendescribed in, for example, WO9425591 for antibodies derived fromCamelidae heavy chain Ig, as well in US20030130496 describing theisolation of single domain fully human antibodies from phage libraries.

Fv and sFv are the only species with intact combining sites that aredevoid of constant regions. Thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins can beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See, for example, AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment also can be a“linear antibody”, for example, as described in U.S. Pat. No. 5,641,870for example. Such linear antibody fragments can be monospecific orbispecific.

In certain embodiments, antibodies of the described invention arebispecific or multi-specific. Bispecific antibodies are antibodies thathave binding specificities for at least two different epitopes.Exemplary bispecific antibodies can bind to two different epitopes of asingle antigen. Other such antibodies can combine a first antigenbinding site with a binding site for a second antigen. Alternatively, ananti-HIV arm can be combined with an arm that binds to a triggeringmolecule on a leukocyte, such as a T-cell receptor molecule (forexample, CD3), or Fc receptors for IgG (Fc gamma R), such as Fc gamma RI(CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD16), so as to focus andlocalize cellular defense mechanisms to the infected cell. Bispecificantibodies also can be used to localize cytotoxic agents to infectedcells. Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (for example, F(ab′)2 bispecific antibodies). Seefor example, WO 96/16673, U.S. Pat. No. 5,837,234, WO98/02463, U.S. Pat.No. 5,821,337, and Mouquet et al., Nature. 467, 591-5 (2010).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, for example,Millstein et al., Nature, 305:537-539 (1983)). Similar procedures aredisclosed in, for example, WO 93/08829, Traunecker et al., EMBO J.,10:3655-3659 (1991) and see also; Mouquet et al., Nature. 467, 591-5(2010). Techniques for generating bispecific antibodies from antibodyfragments also have been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. SeeBrennan et al., Science, 229: 81 (1985).

Typically, the antibodies of described in the invention can be producedusing conventional hybridoma technology or made recombinantly usingvectors and methods available in the art. Human antibodies also can begenerated by in vitro activated B cells (see, for example, U.S. Pat.Nos. 5,567,610 and 5,229,275). General methods in molecular genetics andgenetic engineering useful in the present invention are described in thecurrent editions of Molecular Cloning: A Laboratory Manual (Sambrook, etal., Molecular Cloning: A Laboratory Manual (Fourth Edition) Cold SpringHarbor Lab. press, 2012), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.). Reagents, cloningvectors, and kits for genetic manipulation are available from commercialvendors such as BioRad, Stratagene, Invitrogen, ClonTech andSigma-Aldrich Co.

Human antibodies also can be produced in transgenic animals (forexample, mice) that are capable of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge. See, for example, Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993);U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S.Pat. No. 5,545,807; and WO 97/17852. Such animals can be geneticallyengineered to produce human antibodies comprising a polypeptide of thedescribed invention.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, for example, Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E. coli,thus allowing the facile production of large amounts of these fragments.Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)2 fragments (see, for example, Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)2 fragments can be isolated directly from recombinant host cellculture. Fab and F(ab′)2 fragment with increased in vivo half-lifecomprising a salvage receptor binding epitope residues are described inU.S. Pat. No. 5,869,046. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

Other techniques that are known in the art for the selection of antibodyfragments from libraries using enrichment technologies, including butnot limited to phage display, ribosome display (Hanes and Pluckthun,1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou,et al., 1997, Nature Biotechnology 15: 29-34) and/or yeast display(Kieke, et al., 1997, Protein Engineering 10: 1303-1310) may be utilizedas alternatives to previously discussed technologies to select singlechain antibodies. Single-chain antibodies are selected from a library ofsingle chain antibodies produced directly utilizing filamentous phagetechnology. Phage display technology is known in the art (e.g., seetechnology from Cambridge Antibody Technology (CAT)) as disclosed inU.S. Pat. Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793;5,962,255; 6,140,471; 6,225,447; 6,291650; 6,492,160; 6,521,404;6,544,731; 6,555,313; 6,582,915; 6,593,081, as well as other U.S. familymembers, or applications which rely on priority filing GB 9206318, filed24 May 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14:309-314). Single chain antibodies may also be designed and constructedusing available recombinant DNA technology, such as a DNA amplificationmethod (e.g., PCR), or possibly by using a respective hybridoma cDNA asa template.

Variant antibodies also are included within the scope of the invention.Thus, variants of the sequences recited in the application also areincluded within the scope of the invention. Further variants of theantibody sequences having improved affinity can be obtained usingmethods known in the art and are included within the scope of theinvention. For example, amino acid substitutions can be used to obtainantibodies with further improved affinity. Alternatively, codonoptimization of the nucleotide sequence can be used to improve theefficiency of translation in expression systems for the production ofthe antibody.

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein, orconservative modifications thereof, and wherein the antibodies retainthe desired functional properties of neutralizing multiple HIV-1 viralstrains. As used herein, the term “conservative sequence modifications”refers to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody of the invention by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include

amino acids with basic side chains (e.g., lysine, arginine, histidine),

acidic side chains (e.g., aspartic acid, glutamic acid),

uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan),

nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine),

beta-branched side chains (e.g., threonine, valine, isoleucine) and

aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Thus, one or more amino acid residues within the CDR regions of anantibody of the invention can be replaced with other amino acid residuesfrom the same side chain family and the altered antibody can be testedfor retained function using the functional assays described herein.

Other modifications of the antibody are contemplated herein. Forexample, the antibody can be linked to one of a variety ofnonproteinaceous polymers, for example, polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. The antibody also can be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed in,for example, Remington's Pharmaceutical Sciences, 16th edition, Oslo,A., Ed., (1980).

As disclosed herein, bNAbs are significantly more effective than ART inblocking the establishment of the reservoir when given early in theinfection. One of the key differences between antibodies and ART is thatantibodies can engage a variety of host immune effector pathways by wayof their Fc receptors (Nimmerjahn et al., Nat Rev Immunol, 2008. 8(1):p. 34-47). Consistent with this important difference, the mechanism bywhich antibodies interfere with the establishment of the reservoir isdependent on their ability to bind to Fc receptors.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. An Fc receptor is a protein foundon the surface of certain cells—including, among others, B lymphocytes,follicular dendritic cells, natural killer cells, macrophages,neutrophils, eosinophils, basophils and mast cells—that contribute tothe protective functions of the immune system. Its name is derived fromits binding specificity for the Fc region (fragment crystallizableregion) of an antibody.

Several antibody functions are mediated by Fc receptors. For example, Fcreceptors bind to antibodies that are attached to infected cells orinvading pathogens. Their activity stimulates phagocytic or cytotoxiccells to destroy microbes, or infected cells by antibody-mediatedphagocytosis or antibody-dependent cell-mediated cytotoxicity. It wasalso known in the art that the Fc region of an antibody ensures thateach antibody generates an appropriate immune response for a givenantigen, by binding to a specific class of Fc receptors, and otherimmune molecules, such as complement proteins. FcRs are defined by theirspecificity for immunoglobulin isotypes: Fc receptors for IgG antibodiesare referred to as FcγR, for IgE as FcεFR, for IgA as FcαR and so on.Surface receptors for immunoglobulin G are present in two distinctclasses-those that activate cells upon their crosslinking (“activationFcRs”) and those that inhibit activation upon co-engagement (“inhibitoryFcRs”).

In all mammalian species studied to date, four different classes of IgGFc-receptors have been defined: FcγRI (CD64), FcγRII (CD32), FcγRIII(CDI6) and FcγIV. Whereas FcγRI displays high affinity for the antibodyconstant region and restricted isotype specificity, FcγRII and FcγRIIIhave low affinity for the Fc region of IgG but a broader isotype bindingpattern (Ravetch and Kinet, 1991; Hulett and Hogarth, Adv Immunol 57,1-127 (1994)). FcγRIV is a recently identified receptor, conserved inall mammalian species with intermediate affinity and restricted subclassspecificity (Mechetina et al., Immunogenetics 54, 463-468 (2002); Daviset al., Immunol Rev 190, 123-136 (2002); Nimmerjahn et al., Immunity 23,41-51 (2005)).

Functionally there are two different classes of Fc-receptors: theactivation and the inhibitory receptors, which transmit their signalsvia immunoreceptor tyrosine based activation (ITAM) or inhibitory motifs(ITIM), respectively (Ravetch, in Fundamental Immunology W. E. Paul, Ed.(Lippincott-Raven, Philadelphia, (2003); Ravetch and Lanier, Science290, 84-89 (2000). The paired expression of activating and inhibitorymolecules on the same cell is the key for the generation of a balancedimmune response. Additionally, it has been appreciated that the IgGFc-receptors show significant differences in their affinity forindividual antibody isotypes rendering certain isotypes more strictlyregulated than others (Nimmerjahn et al., 2005).

In one embodiment of the invention, FcR is a native sequence human FcR.In another embodiment, FcR, including human FcR, binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (seereview in Daron, Annu Rev Immunol, 15, 203-234 (1997); FcRs are reviewedin Ravetch and Kinet, Annu Rev Immunol, 9, 457-92 (1991); Capel et al.,Immunomethods, 4, 25-34 (1994); and de Haas et al, J Lab Clin Med, 126,330-41 (1995), Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors inFundemental Immunology, ed William Paul 5th Ed. each of which isincorporated herein by reference).

As used herein, the term “Fc fragment” or “Fc region” is used to definea C-terminal region of an immunoglobulin heavy chain. Such an Fc regionis the tail region of an antibody that interacts with Fc receptors andsome proteins of the complement system. The Fc region may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. A native sequence Fc region comprises an amino acid sequenceidentical to the amino acid sequence of an Fc region found in nature. Avariant Fc region as appreciated by one of ordinary skill in the artcomprises an amino acid sequence which differs from that of a nativesequence Fc region by virtue of at least one “amino acid modification.”

In IgG, IgA and IgD antibody isotypes, the Fc region is composed of twoidentical protein fragments, derived from the second and third constantdomains of the antibody's two heavy chains; IgM and IgE Fc regionscontain three heavy chain constant domains (C_(H) domains 2-4) in eachpolypeptide chain. The Fc regions of IgGs bear a highly conservedN-glycosylation site. Glycosylation of the Fc fragment is important forFc receptor-mediated activity. The N-glycans attached to this site arepredominantly core-fucosylated diantennary structures of the complextype. In addition, small amounts of these N-glycans also bear bisectingGlcNAc and α-2,6 linked sialic acid residues. See, e.g., US20080286819,US20100278808, US20100189714, US 2009004179, 20080206246, 20110150867,and WO2013095966, each of which is incorporated herein by reference.

As the Fc receptor function is involved in HIV-1 latent reservoir, thebNAb antibody of this invention can include antibody variable regionswith the desired binding specificities (antibody-antigen combiningsites) fused to immunoglobulin constant domain sequences. The fusion canbe with an Ig heavy chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. According to some embodiments, thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain bonding, is present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host cell. This providesfor greater flexibility in adjusting the mutual proportions of the threepolypeptide fragments in embodiments when unequal ratios of the threepolypeptide chains used in the construction provide the optimum yield ofthe desired bispecific antibody. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant effect on the yield of the desired chain combination.

C. Inducers

Some embodiments of this invention involve using viral transcriptioninducers. A transcription inducer refers to any agent that can inducethe transcriptional activation of the HIV-1 promoter, or re-activatelatent HIV-1 from the patient viral reservoir directly or indirectly(e.g., facilitating T cell activation by removing an inhibitorypathway). Examples of suitable transcription inducers includevorinostat, an HDAC inhibitor (Contreras et al., J Biol Chem, 2009.284(11): p. 6782-9; Archin et al., AIDS Res Hum Retroviruses, 2009.25(2): p. 207-12; and Archin et al., AIDS, 2009. 23(14): p. 1799-806),I-BET151, a BET bromodomain inhibitor (Boehm et al., Cell Cycle, 2013.12(3): p. 452-62), and αCTLA4, a T-cell inhibitory pathway blocker(Alegre et al., Nat. Rev. Immunol. 2001. p. 220-228 and Krummel et al.,J. Exp. Med. 1995. p. 459-465. See also US 20140128391. For example, theuse of HDAC inhibitors in combination with the bNAbs of this inventioncan be used in disrupting, decreasing, or purging the latently infectedreservoirs in patients, particularly patients undergoing Highly ActiveAntiretroviral Therapy (HAART).

Experiments with indicator cell lines indicate that HDAC and bromodomaininhibitors show synergy when combined with conventional transcriptionalactivators in re-activating HIV-1 in vitro. Consistent with the in vitroexperiments, the combination of these inducers appears to be synergisticin vivo since single inducers had no measurable effect above thebackground controls, while ˜57% of the hu-mice treated with antibodiesplus combination inducers failed to rebound. Irrespective of themechanism, the reservoir of HIV-1 infected cells remaining aftercombination inducer and antibody therapy in hu-mice is significantlydecreased, establishing the principle that the HIV-1 reservoir can bealtered by combination therapy with antibodies and inducers in vivo.

D. Other Anti-Retroviral Agents

The above-described antibodies and viral transcription inducers can usedin combination with one or more anti-retroviral agents for the treatmentof HIV latency and/or infection. See, e.g., US 2010/0166806, US2010/0324034, and US 2012/0203014, which are hereby incorporated intheir entirety.

Compositions according to the present invention may also be administeredin combination with other agents to enhance the biological activity ofsuch agents. Such agents may include any one or more of the standardanti-HIV agents which are known in the art, including, but not limitedto, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine(ddl). Additional agents which have shown anti-HIV effects and may becombined with compositions in accordance to the invention include, forexample, raltegravir, maraviroc, bestatin, human chorionic gonadotropin(hCG), levamisole, estrogen, efavirenz, etravirine, indomethacin,emtricitabine, tenofovir disoproxil fumarate, amprenavir, tipranavir,indinavir, ritonavir, darunavir, enfuvirtide, and gramicidin.

E. Treatment Compositions and Methods

In one embodiment, the present invention provides a compositioncomprising at least one bNAb mentioned above alone or in combinationwith one of the other active agent mentioned above and apharmaceutically acceptable carrier. The composition may include aplurality of the antibodies having the characteristics described hereinin any combination and can further include antibodies neutralizing toHIV as are known in the art.

It is to be understood that compositions can be a single or acombination of antibodies disclosed herein, which can be the same ordifferent, in order to prophylactically or therapeutically treat theprogression of various subtypes of HIV infection. When an antibody oractive agent is administered to an animal or a human, it can be combinedwith one or more pharmaceutically acceptable carriers, excipients oradjuvants as are known to one of ordinary skilled in the art.

Further, with respect to determining the effective level in a patientfor treatment of HIV, in particular, suitable animal models areavailable and have been widely implemented for evaluating the in vivoefficacy against HIV of various therapy protocols. These models includemice, monkeys and cats. Even though these animals are not naturallysusceptible to HIV disease, chimeric mice models (for example, SCID,bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, bonemarrow-ablated BALB/c) reconstituted with human peripheral bloodmononuclear cells (PBMCs), lymph nodes, fetal liver/thymus or othertissues can be infected with lentiviral vector or HIV, and employed asmodels for HIV pathogenesis. Similarly, the simian immune deficiencyvirus (SIV)/monkey model can be employed, as can the feline immunedeficiency virus (FIV)/cat model. The pharmaceutical composition cancontain other pharmaceuticals, in conjunction with a vector according tothe invention, when used to therapeutically treat AIDS. These otherpharmaceuticals can be used in their traditional fashion (i.e., asagents to treat HIV infection).

According to another embodiment, the present invention provides anantibody-based pharmaceutical composition comprising an effective amountof an isolated bNAb of the invention, or an affinity matured version,which provides a prophylactic or therapeutic treatment choice to reducethe latent reservoir and infection of the HIV virus. The pharmaceuticalcompositions of the present invention may be formulated by any number ofstrategies known in the art (e.g., see McGoff and Scher, 2000, SolutionFormulation of Proteins/Peptides: In McNally, E. J., ed. ProteinFormulation and Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158;Akers and Defilippis, 2000, Peptides and Proteins as ParenteralSolutions. In: Pharmaceutical Formulation Development of Peptides andProteins. Philadelphia, Pa.: Talyor and Francis; pp. 145-177; Akers, etal., 2002, Pharm. Biotechnol. 14:47-127). A pharmaceutically acceptablecomposition suitable for patient administration will contain aneffective amount of the bNAb antibody in a formulation which bothretains biological activity while also promoting maximal stabilityduring storage within an acceptable temperature range. Thepharmaceutical compositions can also include, depending on theformulation desired, pharmaceutically acceptable diluents,pharmaceutically acceptable carriers and/or pharmaceutically acceptableexcipients, or any such vehicle commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. The amount of an excipient that is usefulin the pharmaceutical composition or formulation of this invention is anamount that serves to uniformly distribute the antibody throughout thecomposition so that it can be uniformly dispersed when it is to bedelivered to a subject in need thereof. It may serve to dilute theantibody or other active agent to a concentration which provides thedesired beneficial palliative or curative results while at the same timeminimizing any adverse side effects that might occur from too high aconcentration. It may also have a preservative effect. Thus, for anactive ingredient having a high physiological activity, more of theexcipient will be employed. On the other hand, for any activeingredient(s) that exhibit a lower physiological activity, a lesserquantity of the excipient will be employed.

The above described bNAb antibodies and antibody compositions,comprising at least one or a combination of the antibodies describedherein, can be administered for the prophylactic and therapeutictreatment of HIV viral infection.

The composition can be a pharmaceutical composition that contains apharmaceutically acceptable carrier. The term “pharmaceuticalcomposition” refers to the combination of an active agent with acarrier, inert or active, making the composition especially suitable fordiagnostic or therapeutic use in vivo or ex vivo. A “carrier” as usedherein includes pharmaceutically acceptable carriers, excipients, orstabilizers that are nontoxic to the cell or mammal being exposedthereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include, but not limitedto, buffers such as phosphate, citrate, and other organic acids;antioxidants including, but not limited to, ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such as, butnot limited to, serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as, but not limited to, polyvinylpyrrolidone; amino acidssuch as, but not limited to, glycine, glutamine, asparagine, arginine orlysine; monosaccharides, disaccharides, and other carbohydratesincluding, but not limited to, glucose, mannose, or dextrins; chelatingagents such as, but not limited to, EDTA; sugar alcohols such as, butnot limited to, mannitol or sorbitol; salt-forming counterions such as,but not limited to, sodium; and/or nonionic surfactants such as, but notlimited to, TWEEN.; polyethylene glycol (PEG), and PLURONICS.

The term “pharmaceutically acceptable carrier” refers to any of thestandard pharmaceutical carriers, such as a phosphate buffered salinesolution, water, emulsions, and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Apharmaceutically acceptable carrier, after administered to or upon asubject, does not cause undesirable physiological effects. The carrierin the pharmaceutical composition must be “acceptable” also in the sensethat it is compatible with the active ingredient and, preferably,capable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active agent.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, and sodium lauryl sulfate.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human primates (particularly higher primates), dog, rodent (e.g.,mouse or rat), guinea pig, cat, and non-mammals, such as birds,amphibians, reptiles, etc. In a preferred embodiment, the subject is ahuman. In another embodiment, the subject is an experimental animal oranimal suitable as a disease model (such as non-human primates). Asubject to be treated can be identified by standard diagnosingtechniques for the disorder.

According to another embodiment, the present invention provides a methodof reducing or preventing the establishment of a latent reservoir of HIVinfected cells in a subject in need thereof (e.g., a subject infectedwith HIV or at risk of infection with HIV), thereby treating infectionwith a HIV infection, comprising administering to the subject apharmaceutical composition comprising the HIV antibodies disclosedherein. The compositions of the invention can include more than oneantibody having the characteristics disclosed (for example, a pluralityor pool of antibodies). It also can include other HIV neutralizingantibodies and/or active agent known in the art.

Subjects at risk for HIV-related diseases or disorders include patientswho have come into contact with an infected person or who have beenexposed to HIV in some other way. Administration of a prophylactic agentcan occur prior to the manifestation of symptoms characteristic ofHIV-related disease or disorder, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

For in vivo treatment of human and non-human patients, the patient isadministered or provided a pharmaceutical formulation including an HIVantibody of the invention. When used for in vivo therapy, the antibodiesof the invention are administered to the patient in therapeuticallyeffective amounts (i.e., amounts that eliminate or reduce the patient'slatent viral reservoir). The antibodies are administered to a humanpatient, in accord with known methods, such as intravenousadministration, for example, as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies can be administeredparenterally, when possible, at the target cell site, or intravenously.In some embodiments, antibody is administered by intravenous orsubcutaneous administration. Therapeutic compositions of the inventionmay be administered to a patient or subject systemically, parenterally,or locally. The above parameters for assessing successful treatment andimprovement in the disease are readily measurable by routine proceduresfamiliar to a physician.

For parenteral administration, the antibodies may be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable, parenteral vehicle.Examples of such vehicles include, but are not limited, water, saline,Ringer's solution, dextrose solution, and 5% human serum albumin.Nonaqueous vehicles include, but are not limited to, fixed oils andethyl oleate. Liposomes can be used as carriers. The vehicle may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, such as, for example, buffers and preservatives.The antibodies can be formulated in such vehicles at concentrations ofabout 1 mg/ml to 10 mg/ml.

The dose and dosage regimen depends upon a variety of factors readilydetermined by a physician, such as the nature of the infection, forexample, its therapeutic index, the patient, and the patient's history.Generally, a therapeutically effective amount of an antibody isadministered to a patient. In some embodiments, the amount of antibodyadministered is in the range of about 0.1 mg/kg to about 50 mg/kg ofpatient body weight. Depending on the type and severity of theinfection, about 0.1 mg/kg to about 50 mg/kg body weight (for example,about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. The progress ofthis therapy is readily monitored by conventional methods and assays andbased on criteria known to the physician or other persons of skill inthe art. The above parameters for assessing successful treatment andimprovement in the disease are readily measurable by routine proceduresfamiliar to a physician.

Other therapeutic regimens may be combined with the administration ofthe bNAb HIV antibody of the present invention. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active agents simultaneously exert their biological activities.Such combined therapy can result in a synergistic therapeutic effect.The parameters for assessing successful treatment and improvement in thedisease are also readily measurable by routine procedures familiar to aphysician.

The terms “treating” or “treatment” or “alleviation” are usedinterchangeably and refer to both therapeutic treatment and prophylacticor preventative measures; wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. In particular,it refers to administration of a compound or agent to a subject, who hasa disorder (such as an HIV infection), with the purpose to cure,alleviate, relieve, remedy, delay the onset of, prevent, or amelioratethe disorder, the symptom of the disorder, the disease state secondaryto the disorder, or the predisposition toward the disorder.

Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented. A subject or mammal is successfully “treated” for aninfection if, after receiving a therapeutic amount of an antibodyaccording to the methods of the present invention, the patient showsobservable and/or measurable reduction in or absence of one or more ofthe following: reduction in the number of infected cells or absence ofthe infected cells; reduction in the percent of total cells that areinfected; and/or relief to some extent, one or more of the symptomsassociated with the specific infection; reduced morbidity and mortality,and improvement in quality of life issues. The above parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician.

Eliminating the HIV-1 reservoir in chronic infection is key to curingthe disease, but direct measurement of the latent reservoir to evaluatetherapeutic eradication strategies remains difficult (Siliciano et al.,Curr Opin HIV AIDS, 2013. 8(4): p. 318-25). Quantitative viral outgrowthassays and PCR-based assays of integrated DNA yield variable results(Eriksson et al., PLoS Pathog, 2013. 9(2): p. e1003174) in part becausePCR cannot distinguish between inactive and permanently disabledproviruses, and outgrowth assays underestimate reservoir size (Ho etal., Cell, 2013. 155(3): p. 540-51). To that end, the most effective wayto evaluate the reservoir in vivo is to measure viral rebound afterterminating therapy as disclosed in the examples below.

The terms “prevent,” “preventing,” “prevention,” “prophylactictreatment” and the like refer to reducing the probability of developinga disorder or condition in a subject, who does not have, but is at riskof or susceptible to developing a disorder or condition.

A “therapeutically effective amount” refers to the amount of an agentsufficient to effect beneficial or desired results. A therapeuticallyeffective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

Pharmaceutically effective compositions of this invention may beadministered to humans and other animals by a variety of methods thatmay include continuous or intermittent administration. Examples ofmethods of administration may include, but are not limited to, oral,rectal, parenteral, intracisternal, intrasternal, intravaginal,intraperitoneal, topical, transdermal, buccal, or as an oral or nasalspray. Accordingly, the pharmaceutically effective compositions may alsoinclude pharmaceutically acceptable additives, carriers or excipients.Such pharmaceutical compositions may also include the active ingredientsformulated together with one or more non-toxic, pharmaceuticallyacceptable carriers specially formulated for oral administration insolid or liquid form, for parenteral injection or for rectaladministration according to standard methods known in the art.

The term “parenteral” administration refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal,intracisternal, intrasternal, subcutaneous and intraarticular injectionand infusion. Injectable mixtures are known in the art and comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol and the like), vegetableoils (such as olive oil), injectable organic esters (such as ethyloleate) and suitable mixtures thereof.

F. Kits

Another aspect of the invention provides kits. In general, kitsaccording to the present invention comprise an isolated anti-HIV bNAbantibody, a first viral transcription inducer, and a second viraltranscription inducer. Components of the kits can be provided incontainers. The containers are provided in packaged combination in asuitable package, such as a box made of cardboard, glass, plastic,metal, or a combination thereof. Suitable packaging materials forpharmaceutical compositions or reagents are known and widely used in theart, and thus need not be specified herein.

The kits of the invention can comprise any number of additional reagentsor substances that are useful for practicing a method of the invention.Such substances include, but are not limited to: reagents (includingbuffers) for detecting HIV virus or components thereof or HIV-infectedcells in a subject. The kits of the invention can be provided at anytemperature. For example, for storage of kits containing protein-basedagents, it is preferred that they are provided and maintained below 0°C., preferably at or below −20° C., or otherwise in a frozen state.

The kit may further comprise a software package for data analysis of thephysiological status of a subject to be treated, which may includereference profiles for comparison with the relevant test profile. Suchkits may also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such information may bebased on the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like. Kits may also, in some embodiments,be marketed directly to the consumer.

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, unless the context clearly dictates otherwise, between theupper and lower limits of that range is also specifically disclosed.Each smaller range between any stated value or intervening value in astated range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange, and each range where either, neither, or both limits are includedin the smaller ranges is also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The term “about” generally refers to plus or minus 10% of the indicatednumber. For example, “about 10%” may indicate a range of 9% to 11%, and“about 1” may mean from 0.9-1.1. Other meanings of “about” may beapparent from the context, such as rounding off, so, for example “about1” may also mean from 0.5 to 1.4.

Examples Example 1 Materials and Methods

This example describes materials and methods used in Examples 2-5 below.

Mice

NOD Rag1^(−/−)I12rg^(NULL) (NOD.Cg-Rag1^(tmlMom) I12rg^(tmlWjl)/SzJ,NRG) mice were purchased from The Jackson Laboratory. All mice were bredand maintained at the Comparative Bioscience Center of The RockefellerUniversity according to guidelines established by the InstitutionalAnimal Committee. All experiments were performed with authorization fromthe Institutional Review Board and the IACUC at The RockefellerUniversity.

Humanized Mice

Humanized mice were generated as previously described in Klein et al.,Nature, 2012. 492(7427): p. 118-22. Briefly, human fetal livers wereobtained from Advanced Bioscience Resources (ABR). Fetal livers werehomogenized and incubated in HBSS media with 0.1% collagenase IV(Sigma-Aldrich), 40 mM HEPES, 2 mM CaCl2 and 2 U ml⁻¹ DNAase I (Roche)for 30 minutes at 37° C. Hematopoietic Stem Cells (HSCs) were isolatedfrom digested liver using CD34⁺ HSC isolation kit (Stem CellTechnologies). Neonatal NRG mice (1-5 days old) were sublethallyirradiated with 100 cG and injected intrahepatically with 2×10⁵ humanCD34⁺ HSCs 6 h after irradiation.

Mouse Screening for Humanization

Eight or more weeks after HSC injection, mice were screened for thepresence of human lymphocytes in peripheral blood by flow cytometry. 200μl whole blood was collected by facial vein bleed and peripheral bloodmononuclear cells (PBMCs) were isolated by density gradientcentrifugation using Ficoll-Paque Plus (GE Healthcare Life Sciences).PBMCs were stained with antibodies to mouse CD45-PECy7, humanCD45-Pacific Orange, human CD3-Pacific Blue, human CD19-APC, humanCD4-PE, human CD8-FITC, and human CD16-Alexa700 for 25 min at 4° C.Cells were washed and fixed using Cytofix/Cytoperm (BD Biosciences).Flow cytometry analysis was performed with a LSRFortessa (BD) and FlowJosoftware (Tree Star). For each mouse, the percentage of humanlymphocytes [(100×human CD45⁺)/(human CD45⁺+mouse CD45⁺)], termedhuCD45⁺ %, and the percentage of human CD4⁺ T cells (100×human CD45⁺CD3⁺ CD4⁺/human CD45⁺), termed huCD4⁺ %, was calculated. Mice with atleast 10% huCD45⁺ and 10% huCD4⁺ were selected for post-exposureprophylaxis experiments, and infected with two doses of HIV-1_(YU2) (150ng p24) by i.p. injections, 24 hours apart. Pre-treatment viremia wasmeasured at 72-96 hours following the first HIV-1_(YU2) injection, andtreatment was initiated 4 days following the first injection. Forexperiments assessing the effects of bNAbs and inducers on establishedinfections, mice with measurable human CD4⁺ cells by FACS were injectedwith two doses of HIV-1_(YU2) (150 ng), and pre-treatment viremia wasmeasured 14-18 days after the first injection. Mice with plasma viralloads >3000 RNA copies/ml were selected to receive antibody therapy.After five subcutaneous antibody injections (see below), post-treatmentviremias were measured. Only mice with completely suppressed plasmaviremias were selected for further analysis and to receive viralinducers.

Plasma Viral Load Measurements

300-500 μl of whole blood was collected from mice at each time point byfacial vein bleed. Whole blood was spun at 300 g for 10 minutes toseparate plasma from the cellular fraction. Total RNA was extracted from100 μl plasma using QIAmp MinElute Virus Spin Kit (Qiagen) incombination with RNase-free DNase (Qiagen), eluted in a 50 μl volume.HIV-1 RNA was quantified by qRT-PCR. The reaction mixture was preparedusing TaqMan RNA-to-Ct 1-Step kit (Applied Biosystems), with 20 μl ofeluted RNA, and a sequence specific probe targeting a conserved regionof the HIV-1 pol gene (/HEX/5′-CCCACCAACARGCRGCCT TAACTG-3′/ZenDQ, HXB2nt 4603 to 4626, SEQ ID No: 44) (Integrated DNA Technologies). Forwardand reverse primer sequences were 5′-TAATGGCAGCAATTTCACCA-3′ (HXB2 nt4577-4596, SEQ ID No: 45) and 5′-GAATGCCAAATTCCTGCTTGA-3′ (HXB2 nt 4633to 4653, SEQ ID No: 46), respectively. Cycle threshold (Ct) values werecalibrated using standard samples with known amounts of absolute viralRNA copies. The quantitation limit was previously determined to be 800copies/ml (Klein et al., Nature, 2012.492(7427): p. 118-22).

Gp120 Sequencing

Gp120 cloning and sequencing was performed as previously described(Klein et al., 2012, HIV therapy by a combination of broadlyneutralizing antibodies in humanized mice. In Nature (Nature PublishingGroup), pp. 118-122). Briefly, cDNA was synthesized from viral RNA usingSuperScript III reverse transcriptase (Invitrogen Life Technologies).cDNA was amplified with Expand Long Template PCR System (Roche) withnested PCR. Primers for the first round of PCR were5′-GGCTTAGGCATCTCCTATGGCAGGAAGAA-3′ and5′-GGTGTGTAGTTCTGCCAATCAGGGAAGWAGCCTTGTG-3′ (SEQ ID Nos: 47 and 48).Primers for the second round of PCR were5′-TAGAAAGAGCAGAAGACAGTGGCAATGA-3′ and5′-TCATCAATGGTGGTGATGATGATGTTTTTCTCTCTGCACCACTCTTCT-3′(SEQ ID Nos: 49and 50). Gel-purified PCR amplicons were ligated into pCR4-TOPO(Invitrogen) and transformed into One Shot TOP10 cells. Individualcolonies were sequenced using M13F and M13R primers. Sequences werealigned to gp120_(YU2) (accession number M93258) and analyzed formutations using Los Alamos Highlighter tool(hiv.lanl.gov/content/sequence/HIGHLIGHT/HIGHLIGHT_XYPLOT/highlighter.html).

Cell-Associated HIV-1 RNA

The cellular fraction of whole blood was resuspended in 400 μl PBS andPBMCs were isolated by density gradient centrifugation as describedabove. Lymphocytes were split into two samples, one for cell-associatedHIV-1 RNA measurements, and one for cell-associated HIV-1 DNAmeasurements. Cell-associated RNA was extracted and quantified by thesame procedures as described above for plasma viral RNA. The lower limitof detection was determined to be 10 copies viral RNA per qRT-PCRreaction. Cell-associated HIV-1 RNA is reported as the ratio of HIV-1RNA copies per sample to CCR5 genomic DNA copies per equivalent samplemeasured in DNA extract. For terminal point measurements, spleen tissuewas isolated, homogenized, and filtered through 40 μm mesh. Splenocyteswere used to isolate HIV-1 RNA as described above.

Cell-Associated HIV-1 DNA

PBMCs were isolated from whole blood as described above. Splenocyteswere isolated from spleen as described above. Total DNA was extractedusing QIAmp DNA Blood Mini Kit (Qiagen) and eluted in 80 μl volume.Purified DNA was quantified for HIV-1 DNA by qPCR using the primers andprobe for HIV-1 RNA quantification mentioned above. Genomic human CCR5DNA was quantified with primers 5′-GTTGGACCAAGCTATGCAGGT-3′ (forward,SEQ ID No: 51) and 5′-AGAAGCGTTTGGCAATGTGC-3′ (reverse, SEQ ID No: 52),and the sequence-specificprobe/HEX/5′-TTGGGATGACGCACTGCTGCATCAACCCCA-3′/ZenDQ (SEQ ID No: 53).All qPCR reactions contained 25 μl AmpliTaq Gold PCR master mix (AppliedBiosystems), in 50 μl reaction volume. Reaction mixtures were aspreviously described (Horwitz et al., Proc Natl Acad Sci USA, 2013.110(41): p. 16538-43). HIV-1 DNA is reported as copies per sample toCCR5 genomic copies per equivalent sample.

Terminal Graft

The presence of human lymphocytes at the terminal point was quantifiedfrom the spleen and PBMCs by flow cytometry. Isolation of PBMCs andsplenocytes were as described above. Staining procedures were asdescribed above.

Antibody Concentrations

Plasma levels of passively administered antibodies were quantified bytwo independent methods. gp120-specific ELISA was as previouslydescribed (Klein et al., Nature, 2012. 492(7427): p. 118-22), using10-1074 and 3BNC117 monoclonal antibodies as standard controls. Thedetection limit was 0.05 μg/ml. Because PG16 does not bind gp120, andendogenously produced gp120-reactive antibodies could confound the ELISAmeasurement, plasma antibody levels were also quantified by TZM-blneutralization using the Tier 2 envelopes 3301.v1.c24 and YU2. A mixturewith known amounts of 3BNC117, 10-1074, and PG16 was used as standardfor calibration.

Day of Viral Rebound and Antibody Level at Rebound

Plasma viremias immediately preceding and following viral rebound wereplotted on a semi-log-y-axis versus days post initial antibody injection(x-axis) for each individual mouse. The linear portion of viremia wasfit to a line by least-squares linear regression. The day that viremiacrossed the 800 copies/ml quantitation limit, termed rebound day, wascalculated from the viremia fit. The antibody concentrations (asdetermined by TZM-bl neutralization) spanning before and after viralrebound were plotted on a semi-log-y-axis versus days post initialantibody injection. The linear portion of antibody concentrations wasfit to a line by least-squares linear regression, and the antibodyconcentration on the rebound day was calculated from the fit.

Anti-Retroviral Therapy

Individual tablets of tenofovir disproxil-fumarate (TDF; GileadSciences), emtricitabine (FTC; Gilead Sciences), and raltegravir (RAL;Merck) were crushed into fine powder and manufactured with TestDiet 5B1Qfeed (Modified LabDiet 5058 with 0.12% amoxicillin) into ½″ irradiatedpellets. Final concentrations of ART drugs in the food were 720 mg/kgTFV, 520 mg/kg FTC, and 4800 mg/kg RAL. Doses were chosen based onsuppression of viremia in humanized mice as previously published (Dentonet al., J Virol, 2012. 86(1): p. 630-4 and Nischang et al., PLoS ONE2012. p. e38853), and by pharmacokinetic analysis of these drugs inhumanized mice (unpublished, Speck Laboratory). To test potentialtoxicity, or reduced preference for drug-supplemented food, mice wereweighed daily on normal diet, then switched to ART feed and weigheddaily. There were no visible signs of toxicity and mice maintained theirweights. Assuming mice weigh 25 grams and eat 4 grams of food per day,the drug doses correspond to 2.88 mg/kg TFV, 83 mg/kg FTC, and 768 mg/kgRAL daily.

Antibody Therapy

Plasmids encoding 10-1074 or PG16 heavy- and light-chain Ig genes weretransfected into HEK 293E cells. Antibodies were isolated fromtissue-culture supernatant using Protein G Sepharose 4 Fast-Flow (GEHealthcare). Antibodies were then buffer-exchanged into PBS andsterile-filtered using Ultrafree-CL centrifugal filters (0.22 μm;Millipore). Endotoxin was removed from antibody preparations usingTriton X-114 (Sigma-Aldrich) as previously described (Aida et al., JImmunol Methods, 1990. 132(2): p. 191-5), and antibodies wereconcentrated to 10 mg/ml. Sterile, endotoxin-free 3BNC117 (20 mg/ml) wasobtained from CellDex Therapeutics. All antibodies were injectedsubcutaneously as described.

Inducers

Vorinostat (Selleckchem) was suspended in sterile water or sterile waterplus 0.5% methylcellulose, 0.1% Tween (v/v) and administered by oralgavage at doses of 60 mg/kg (Krejsgaard et al., 2010 Experimentaldermatology 19, 1096-1102). For each mouse, three total doses wereadministered, spaced 48 hours apart. 100 μg doses of αCTLA4 wereinjected intraperitoneally (i.p.). Three total doses were administered,spaced 48 hours apart. I-BET was obtained from GlaxoSmithKline anddissolved in 10% beta-cyclodextrin, 5% DMSO in 0.9% saline and injecteddaily for 14 days at doses of 30 mg/kg (Dawson et al., 2011 Nature 478,529-533.).

Statistical Analysis

Statistical Analyses were Performed Using GraphPad Prism 6.0 for Mac OSX.

Example 2 Post Exposure Prophylaxis with bNAbs

The ART-resistant reservoir is established early in infection asevidenced by post-exposure prophylaxis experiments in humans andmacaques. Post-exposure prophylaxis with ART or previous-generationbNAbs is only effective when administered within 24 hours of intravenousexposure. (Lifson et al., Journal of Virology 2000. p. 2584-2593; Wadeet al., N Engl J Med, 1998. 339(20): p. 1409-14; Tsai, et al., Journalof Virology 1998. p. 4265; Tsai et al., Science 1995. p. 1-3; Landovitzet al., in N Engl J Med 2009. p. 1-8; Nishimura et al., Proc Natl AcadSci USA, 2003. 100(25): p. 15131-6; and Ferrantelli et al., Virology,2007. 358(1): p. 69-78). To determine if the current generation of morepotent bNAbs can abort the establishment of a latent HIV-1 reservoir atlater time points, post-exposure prophylaxis experiments were performedin humanized mice (FIG. 1A).

Mice were infected with HIV-1_(YU2) (150 ng) by intraperitonealinjection, and treated with either ART (raltegravir, emtricitabine,tenofovir) (Denton et al., J Virol, 2012. 86(1): p. 630-4 and Nischanget al., PLoS ONE 2012. p. e38853) or a tri-mix of bNAbs (3BNC117,10-1074, and PG16) (Horwitz et al., Proc Natl Acad Sci USA, 2013.110(41): p. 16538-43) 4 or 8 days after infection when viremia wasalready detectable in 51 of 70 mice. Plasma viremia varied fromundetectable to 2.70×10⁶ viral RNA copies/ml at 4 days after infection(FIGS. 1B-E and 6). In the absence of therapy, 14 out of 15 mice in thecontrol group developed sustained plasma viremia ranging from 2.48×10³to 4.19×10⁶ copies/ml (FIG. 1B).

Doses of ART and antibodies were chosen on the basis of theirtherapeutic efficacy in chronic HIV-1 infection in hu-mice (Klein etal., Nature, 2012. 492(7427): p. 118-22; Horwitz et al., Proc Natl AcadSci USA, 2013. 110(41): p. 16538-43; Denton et al., J Virol, 2012.86(1): p. 630-4; and Nischang et al., PLoS ONE 2012. p. e38853). ART wasadministered in the food for 32-39 days starting 4 days after infection(Denton et al., J Virol, 2012. 86(1): p. 630-4; and Nischang et al.,PLoS ONE 2012. p. e38853). Antibodies were administered subcutaneouslywith a loading dose of 3 mg per mouse, and 3-5 subsequent doses of 1.5mg each, spaced 3-4 days apart (FIG. 1A). Consistent with human andmacaque studies, 18 of 22 mice treated with ART showed viremia after ARTtermination, demonstrating that this form of therapy is relativelyineffective at preventing reservoir development in hu-mice whenadministered 4 days after infection (FIG. 1C). Among the 18 viremicmice, viremia was first detected 28 to 84 days after ART termination(FIGS. 1C and 6). In contrast, 10 of 21 hu-mice treated with antibodies4 days after infection showed viremia by the terminal point (p=0.027),and for 9 of these 10 viremic mice, the first detectable viremiaoccurred 74-107 days after the last antibody injection (FIGS. 1D and 6).However, bNAb treatment after 8 days was far less effective, resultingin viremia in 10 of the 11 treated mice 44-58 days after the lastantibody injection (FIG. 1E).

Mice in the early treatment group that failed to show detectable plasmaviremia were further examined for the presence of human CD4⁺ T cells andcell-associated HIV-1 RNA and DNA in the spleen. It was found that micethat failed to develop sustained plasma viremia showed CD4⁺ T celllevels that were similar to infected controls. Therefore differences inCD4⁺ T cell levels are unlikely to account for the observed differencesbetween viremic and aviremic mice (FIG. 1F). Moreover, T cell-associatedHIV-1 RNA levels were consistent with plasma viral loads, with mice thatremained aviremic having either undetectable or lower cell-associatedHIV-1 RNA than mice that developed sustained viremia (FIG. 1G).

Cell-associated viral DNA was measured as an imperfect surrogate of theHIV-1 reservoir. HIV-1 DNA is thought to overestimate the reservoirbecause it fails to exclude damaged or incomplete viral sequences thatcannot be reactivated. In addition, the overall number cells assayed inmice is limited and therefore the assay is not very sensitive.Nevertheless HIV-1 DNA measurements were found to be consistent witheach mouse's rebound status (FIG. 1H). It was concluded that bNAbs caninterfere with the establishment of the latent HIV-1 reservoir inhu-mice.

The above results indicate that bNAbs differ from ART in that they canprevent establishment of the latent HIV-1 reservoir in hu-mice at a timewhen ART is significantly less effective.

Example 3 Fc Receptor Binding is Required for bNAb Activity

To determine if the efficacy of bNAbs is dependent on the antibodies'ability to engage components of the immune system through their Fcdomains, the day 4 post-exposure prophylaxis experiments were repeatedusing the same tri-mix of bNAbs carrying Fc region mutations thatabrogate both human and mouse Fc-receptor binding (G236R/L328R; GRLR,herein referred to as FcR^(null)) (Horton et al., 2010, Blood 116,3004-3012). Despite equivalent neutralizing activity in TZM-bl assays(Pietzsch et al., Proc Natl Acad Sci USA, 2012. 109(39), Fcr^(null)antibodies were far less potent than controls in vivo (FIGS. 2 and 7).Mice treated with Fcr^(null) tri-mix initially suppressed viremia at thesame rate as the wild type antibody-treated mice (FIG. 2A). However 9 of15 mice receiving post exposure prophylaxis with the Fcr^(null) tri-mixshowed viral rebound by 44 days after the last antibody injection. Incontrast, 44 days after the last injection of control antibodies, only 1of 21 mice showed rebound viremia (p=0.0004). The loss of in vivoactivity of the Fcr^(null) tri-mix was not due to lower antibody levelsin the Fcr^(null) injected mice. In fact, antibody levels at the time ofviral rebound were ˜50-fold higher for mice receiving Fcr^(null) tri-mixcompared to wild-type tri-mix (p=0.0035, FIG. 2C). It was concluded thateffective post-exposure prophylaxis by bNAbs requires engagement ofFc-receptors.

The escape variants to the individual bNAbs in the tri-mix used in theseexperiments have been documented extensively (Horwitz et al., Proc NatlAcad Sci USA, 2013. 110(41): p. 16538-43, and Klein et al., Nature,2012. 492(7427): p. 118-22). However, inventors have never observedHIV-1 escape by mutation to the bNAb tri-mix. Rather viral rebound isusually due to a drop in antibody concentrations to sub-therapeuticlevels (Horwitz et al., Proc Natl Acad Sci USA, 2013. 110(41): p.16538-43, and Klein et al., Nature, 2012. 492(7427): p. 118-22). Becausemice receiving Fcr^(null) tri-mix showed viral rebound in the presenceof antibody concentrations far higher than the therapeutic threshold forwild-type antibodies (FIG. 2C), gp120 from 9 of the rebounding mice werecloned and sequenced to examine the mechanism for viral breakthrough inthe presence of Fcr^(null) tri-mix (FIG. 2D). Among all 40 clonessequenced, not a single clone had the triple combination of signaturemutations that confer escape to the antibody-tri-mix. It was concludedthat viral rebound in Fcr^(null) tri-mix treated mice is notattributable to antibody escape, but rather reduced antibody potency.Thus, Fcr^(null) mutant antibodies, which cannot engage Fc receptors,are less active in suppressing infection than their wild typecounterparts.

These results indicate that effective post-exposure prophylaxis by bNAbsrequires engagement of Fc-receptors.

Example 4 Combination Therapy with bNAbs and Inducers

A small number (˜15%) of chronically infected hu-mice and macaquestreated with antibodies fail to show rebound viremia after therapy isdiscontinued (Barouch et al., Nature, 2013. 503(7475): p. 224-8; Horwitzet al., Proc Natl Acad Sci USA, 2013. 110(41): p. 16538-43; Klein etal., Nature 2012, p. 118-122; and Shingai et al., Nature, 2013.503(7475): p. 277-80). This suggests that antibodies may be able todecrease the size of the reservoir, or interfere with its maintenance,in established infections. To determine whether agents that induce viraltranscription from latently infected cells can enhance this effectantibody therapy was combined with viral inducers (FIGS. 3A and 8).

Hu-mice with established HIV-1_(YU2) infections (viremia ranging from4.70×10³-7.96×10⁵ copies/ml at 2-3 weeks after infection, FIGS. 3B-E)were treated with tri-mix bNAbs. When plasma viremia dropped belowdetection, they were co-administered a viral inducer for 5-14 days, andmonitored for viral rebound for an additional 47-85 days. The inducerstested were vorinostat, an HDAC inhibitor (Archin et al., AIDS Res HumRetroviruses, 2009. 25(2): p. 207-12; Archin et al., AIDS, 2009. 23(14):p. 1799-806; and Contreras et al., J Biol Chem, 2009. 284(11): p.6782-9), I-BET151, a BET protein inhibitor (Boehm et al., Cell Cycle,2013. 12(3): p. 452-62), and αCTLA4, a T-cell inhibitory pathway blocker(Alegre et al., Nat. Rev. Immunol 2001. p. 220-228, and Krummel et al.,J. Exp. Med. 1995. p. 459-465). They were selected because of theirdocumented abilities to induce HIV-1 transcription in vitro, as well astheir safety and established pharmacokinetic properties in mice(Krejsgaard et al., Exp Dermatol, 2010. 19(12): p. 1096-102; Kwon etal., Proc Natl Acad Sci USA, 1997. 94(15): p. 8099-103; and Nicodeme etal., Nature, 2010. 468(7327): p. 1119-23).

Hu-mice receiving antibodies plus vorinostat showed no significantdifferences in viral rebound compared to hu-mice receiving antibodyalone (FIGS. 3B, 3C and 8). The same result was seen for hu-mice treatedwith antibodies plus I-BET151 or αCTLA4 (FIGS. 3D, 3E and 8). All 10mice that received antibody therapy plus vorinostat showed viral reboundwhen the antibody dropped below therapeutic levels. Of 12 mice thatreceived antibody therapy plus I-BET151, 11 had viral rebound, and 10 of11 mice that received antibody plus αCTLA4 showed viral rebound. Intotal, of 33 mice that received antibody plus a single inducer, 31showed viral rebound. In comparison, of 25 mice that received antibodytherapy alone, 22 rebounded after the level of passively administeredantibody decayed below the therapeutic threshold (p=0.64).

Of the three inducers tested, vorinostat is the only one that has alsobeen studied in HIV-1 infected humans. Treatment with vorinostat plusART resulted in a transient increase in resting CD4⁺ T cell-associatedHIV-1 RNA, but no change in plasma viremia, or the frequency ofreplication-competent HIV-1 within resting CD4⁺ T cells (Archin et al.,Nature, 2012. 487(7408): p. 482-5 and Archin et al., J. Infect. Dis.2014. p. 1-26). The above results in hu-mice are consistent with thesefindings, and extend them to additional candidate inducers,demonstrating that administration of a single inducer has no significanteffect on the ability of the latent reservoir to produce reboundviremia.

Example 5 Combination Therapy with Multiple Inducers

In this example, assays were carried out to determine whether acombination of inducers might be more effective than a single inducer.More specifically, all three inducers mentioned above were administeredsimultaneously.

It was found that, in the absence of antibody therapy, the combinationof all three inducers was not measurably toxic and did not abort ornoticeably alter active infection (FIG. 6). 23 mice that initiallysuppressed viremia on antibody therapy were treated with the inducercombination and followed for 62-105 days after the last antibodyinjection (FIG. 4A). Only 10 of the 23 showed viral rebound, and theremaining 57% of the mice failed to rebound, a significant decrease inrebound frequency compared to antibody alone (p=0.0018), or antibodyplus single inducers (p=0.0001) (FIG. 4B).

Importantly, when compared to antibody alone, neither single inducer norcombination inducers measurably altered the frequency of CD4⁺ T cellsremaining at the end of the experiment (FIG. 5A). Additionally, spleenT-cell associated viral RNA reflected plasma viral RNA levels at thetime the experiment was terminated in that it was largely undetectablein mice that failed to rebound (FIG. 5B).

Finally, when compared to controls, hu-mice that failed to rebound aftercombination antibody and inducer therapy showed similar initial plasmaviremias to mice that rebounded across all experimental groups (FIG.5C). Therefore, neither initial viremia levels, nor CD4⁺ T cell levelscan account for the differences between the experimental groups.

To determine if antibody persistence or premature termination accountedfor differing viral rebound outcomes, antibody levels at the time ofrebound and at the terminal point were calculated. The average plasmaantibody concentration at the time of viral rebound in the 59 reboundingmice was 2.97 μg/ml (FIG. 5D). Since the antibody concentrations decayedto 2.97 μg/ml at different rates in individual mice, the number of daysthat elapsed from when each individual mouse's antibody levels reached2.97 μg/ml to when the mouse showed rebound viremia were calculated. 50of 59 mice rebounded within 10 days (FIG. 5E). Of the 18 non-reboundingmice, the average antibody concentration at the terminal point was 0.44μg/ml, with 15 out of 18 mice having antibody concentrations less than2.97 μg/ml. Furthermore, in non-rebounding mice, an average of 20.2 dayselapsed from the time antibody concentrations reached 2.97 μg/ml totermination (FIG. 5F). Thus, failure to rebound cannot be explained byantibody persistence or premature termination.

Finally, inventors could not detect viral DNA at the terminal point inthe majority of mice that did not rebound, whereas the majority of micethat did rebound had detectable HIV-1 DNA, with an average of 0.09copies per T cell (FIG. 5G). This suggests that combining vorinostat,I-BET151 and αCTLA4 with immunotherapy decreases rebound viremia inhu-mice.

In the above example, hu-mice resembled infected humans in that theycontain human cells that are infected with authentic HIV-1 (Hatziioannouet al., Nat Rev Microbiol, 2012. 10(12): p. 852-67 and Brehm et al., JInfect Dis, 2013. 208 Suppl 2: p. S125-30). In addition, the kinetics ofviral rebound in hu-mice after suppression of viremia with ART resemblesinfected humans (Horwitz et al., Proc Natl Acad Sci USA, 2013. 110(41):p. 16538-43 and Nischang et al., PLoS ONE 2012. p. e38853). The macaquemodel is valuable because it represents an immunologically intact hostthat may also harbor reservoirs not found in the mice. However, theinfection in macaques involves non-human primate cells and SHIV or SIV,both of which differ significantly from HIV-1. While neither of the twomodel systems is entirely faithful, they have produced very similarresults in both therapy and prevention experiments to date (West et al.,Cell, 2014. 156(4): p. 633-48).

One of the strategies proposed to eliminate latent viruses involvesinducing their expression under the cover of ART. In theory, this wouldkill infected cells while preventing the spread of infection (Deeks, S.G., Nature, 2012. 487(7408): p. 439-40). In vitro experiments indicatethat silent proviruses can in fact be induced to become active (Ho etal., Cell, 2013. 155(3): p. 540-51 and Bullen et al., Nature, 2014, p.1-6), but neither infected neither humans nor hu-mice show a measurablechange in the reservoir after therapy with a single inducer and ART(Archin et al., J. Infect. Dis. 2014. p. 1-26) or bNAbs. The resultshere establish the principle that only a combination of inducers andbNAbs can have a significant impact on the viral reservoir in vivo.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference in their entireties.

1. A method for decreasing the size of or preventing the establishmentof a latent reservoir of HIV infected cells in a subject in needthereof, comprising administering to said subject a therapeuticallyeffective amount of an isolated anti-HIV antibody, and administering tosaid subject two or more viral transcription inducers in effectiveamounts to induce transcription of an HIV provirus in said cells.
 2. Themethod of claim 1, wherein the antibody is a human antibody, a humanizedantibody, or a chimeric antibody.
 3. The method of claim 1, wherein theantibody is antibody 3BNC117, 10-1074, or PG16.
 4. The method of claim3, wherein two or more of said antibodies 3BNC117, 10-1074, and PG16 areadministered to said subject.
 5. The method of claim 1, wherein one,two, or more of antibodies listed in Table 1 are administered to saidsubject.
 6. The method of claim 1, wherein the transcription inducersare selected from the group consisting of vorinostat, an HDAC inhibitor,I-BET151, a BET bromodomain inhibitor, αCTLA4, and a T-cell inhibitorypathway blocker.
 7. The method of claim 1, wherein said cells compriseCD4⁺ T cells.
 8. The method of claim 1, wherein the antibody orantibodies are administered to the subject within about 96 hours afterexposure to HIV.
 9. The method of claim 1, further comprisingadministering to said subject an antiviral agent.
 10. The method ofclaim 8, wherein the antiviral agent is selected from the groupconsisting of a non-nucleoside reverse transcriptase inhibitor, aprotease inhibitor, an entry or fusion inhibitor, and an integraseinhibitor.
 11. A kit comprising an isolated anti-HIV antibody, a firstviral transcription inducer, and a second viral transcription inducer.12. The kit of claim 11, wherein the antibody is antibody 3BNC117,10-1074, or PG16.
 13. The kit of claim 11, wherein kit comprises two ormore of said antibodies 3BNC117, 10-1074, and PG16.
 14. The kit of claim11, wherein the antibody is a human antibody, a humanized antibody, or achimeric antibody.
 15. The kit of claim 11, wherein the transcriptioninducers are selected from the group consisting of vorinostat, an HDACinhibitor, I-BET151, a BET bromodomain inhibitor, αCTLA4, and a T-cellinhibitory pathway blocker.
 16. The kit of claim 11, wherein the firstviral transcription inducer and the second viral transcription inducerare in one pharmaceutical composition or in two separate pharmaceuticalcompositions.
 17. The kit of claim 11, further comprising an antiviralagent.
 18. The kit of claim 17, wherein the antiviral agent is oneselected from the group consisting of a non-nucleoside reversetranscriptase inhibitor, a protease inhibitor, an entry or fusioninhibitor, and an integrase inhibitor.
 19. A method for preventing theestablishment of a latent reservoir of HIV infected cells in a subjectin need thereof, comprising administering to said subject atherapeutically effective amount of an isolated anti-HIV antibody. 20.The method of claim 19, wherein the antibody is antibody 3BNC117,10-1074, or PG16.
 21. The method of claim 19, wherein the antibody is ahuman antibody, a humanized antibody, or a chimeric antibody.
 22. Themethod of claim 20, wherein two or more of said antibodies 3BNC117,10-1074, and PG16 are administered to said subject.
 23. The method ofclaim 19, wherein the antibody or antibodies are administered to thesubject within about 96 hours after exposure to HIV.